11-Deoxy anthracycline antibiotics, their preparation and use

ABSTRACT

Anthracycline antibiotics of the formula: ##STR1## wherein R is --CO--CH 2  OH, --CHOH--CH 3 , --CO--CH 3  or --CH 2  --CH 3  having antitumor and antibacterial activity are prepared by culturing a new mutant strain of microorganism designated as Micromonospora peucetica sp. nova and which has been deposited with the American Type Culture Collection under number No. 31366 ATCC.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to anthracycline antibiotics which are 11-deoxy analogues of the known anthracycline antibiotics daunomycin and adriamycin*, their aglycones, methods of preparing same, the use thereof, a new strain of microorganism used to make said antibiotics and a method of making the new strain of microorganism.

2. The Prior Art

Daunomycin and adriamycin are known antitumor antibiotics of the anthracycline series. Their aglycones are, of course, also known.

The microorganism, Streptomyces peucetius var. caesius, is also known; see, Arcamone et al., Biotechnol. Biogen., 1969, X1, 1101-1110.

SUMMARY OF THE INVENTION

The invention provides, in one aspect thereof, a new class of anthracycline antibiotics, hereinafter designated as glycosides A, B, C and D, which are 11-deoxy derivatives of daunomycin and adriamycin, as well as their respective aglycones. The new 11-deoxy derivatives have the following formula: ##STR2## wherein R is --COCH₂ OH(I), --CHOH--CH₃ (II), --CO--CH₃ (III) or --CH₂ --CH₃ (IV).

Glycoside A has the formula (I) and is 11-deoxyadriamycin;

Glycoside B has the formula (II) and is 11-deoxy-13-dihydrodaunomycin;

Glycoside C has the formula (III) and is 11-deoxydaunomycin; and

Glycoside D has the formula (IV) and is 11-deoxy-13-deoxo-daunomycin.

The respective agylcones which are obtained from the glycosides by aqueous acid hydrolysis to remove the sugar moiety, i.e., daunosamine have the formula: ##STR3## wherein R is --COCH₂ OH(V), --CHOH--CH₃ (VI), --CO--CH₃ (VII) or --CH₂ --CH₃ (VIII).

The aglycone of glycoside A has the formula (V) and is 11-deoxy-adriamycinone;

The aglycone of glycoside B has the formula (VI) and is 11-deoxy-13-dihydro-daunomycinone;

The aglycone of glycoside C has the formula (VII) and is 11-deoxy-daunomycinone; and

The aglycone of glycoside D has the formula (VIII) and is 11deoxy-13-deoxo-daunomycinone.

In another aspect, the invention provides methods of preparing the glycosides A, B, C and D which method comprises culturing a new mutant strain of the microorganism Streptomyces peucetius var. caesius which produces the glycosides A, B, C and D, and recovering same from the culture medium.

In yet another aspect, the invention provides a method for producing the new mutant strain of said microorganism which comprises subjecting same to a mutagenic treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

In still another aspect, the invention provides said new mutant strain of the microorganism which has been designated number B211 F.I.* of the Farmitalia Collection of Microorganisms. This new mutant strain has been demonstrated to be a new species of the genus Micromonospora. It has also been designated as Micromonospora peucetica sp. nova.

In still another aspect, the invention provides pharmaceutical compositions comprising said glycosides A, B, C or D in combination with an inert carrier therefor.

Finally, the invention provides methods of treating certain mammalian tumors (L 1210 and P 388 leukemia) and bacterial infections using the new glycosides.

By way of summary then, the present invention includes the new anthracycline glycoside antibiotics designated A, B, C and D, both in dilute form as crude concentrates and in pure crystalline form, and the corresponding aglycones. The invention also includes processes for the production of these compounds by fermentation of a mutant strain of Streptomyces peucetius var. caesius, and their recovery, concentration, purification and salt preparation.

The new anthracycline glycosides display antitumor and antibacterial activity. More particularly, glycosides A and C are useful as antitumor agents in experimental animals.

The new microorganism used in the invention is obtained by a mutagenic treatment with N-methyl-N'-nitro-N-nitrosoguanidine of Streptomyces peucetius var. caesius. As in the case of many antibiotic-producing cultures, fermentation of strain B211 F.I. results in the production of a mixture or a complex of components. Four bioactive anthracycline components the glycosides A, B, C and D, have been separated from that complex.

The present anthracycline glycosides all contain daunosamine (3-amino-2,3,6-trideoxy-L-lyxo-hexose), which is the amino sugar component of daunomycin and adriamycin (F. Arcamone, G. Cassinelli, P. Orezzi, G. Franceschi and R. Mondelli, J.Am.Chem.Soc., 86, 5335, 1964 and U.S. Pat. No. 3,590,028). The structures of the new glycosides were determined by analysis of their infrared, ultraviolet, visible, mass and magnetic resonance spectra and are all in agreement with the chemical and physical data provided hereinbelow.

The new anthracycline glycoside complex and its individual components form salts with both acids and bases, and pharmaceutically acceptable salts of the complex and its components are also included within the scope of the invention. Examples of such pharmaceutically acceptable salts include salts with acids such as hydrochloric, sulphuric, nitric, and phosphoric, and with metallic cations, for example, alkali metal or alkaline earth metal cations such as sodium, potassium, calcium and magnesium, and also with other cations such as trivalent iron cations.

DETAILED DESCRIPTION OF THE INVENTION The Microorganism

Microscopic properties of strain B 211 F.I.

The spores of B 211 F.I. are of the following size: 0.9-1.1×1.1-1.6μ. They are borne singly or very frequently in pairs, and only rarely as clusters, terminally on short sporophors arising monopodially as branches on long, randomly produced hyphae 0.5-0.9μ in thickness. Rarely are sessile spores observed. No polymorphic forms are produced and aerial mycelium is absent.

Macroscopic properties of strain B 211 F.I.

The cultural characteristics of strain B 211 F.I. are given below in Table 1. Growth of the microorganism is generally good on organic media, not quite as good on synthetic media; on the former, it is generally raised and ridged, rather moist but non-slimy in appearance.

The sporulation layer turns the orange-terra cotta colored vegetative mycelium brown to almost black ageing. No soluble pigment is generally produced. No growth is observed at temperatures above 40° C. The physiological and biochemical properties of strain B 211 F.I. are given below in Table 2.

Identification and classification of strain B 211 F.I.

The strain B 211 F.I. is clearly referrable to the genus Micromonospora Orskov (Orskov, J. "Investigations into the Morphology of the Ray Fungi", Levin and Maunksgaard, Copenhagen, 1923, page 171) of the order Actinomycetales, on the basis of its morphological and cultural characteristics. However, careful comparative examination of the characteristic described and reported by Waksman, S. A. ("The Actinomycetes" Vol. 2-1961, Williams and Wilkins Co. Baltimore and "The Actinomycetes; A Summary of Current Knowledge", 1967, Ronald Press Co.), Luedemann and Brodsky (1963) and Luedemann, G. ("Micromonospora Taxonomy" Advances in Applied Microbiology, 1970, 11, 101-133) for known species belonging to this genus, with those shown by strain B 211 F.I. did not enable us to identify the present strain with any of the named species listed for the above-mentioned genus. Because of its peculiar carbohydrate utilization pattern, and because it produces new anthracyclines, it may be concluded that strain B 211 F.I. differs from all previously described species of this genus, and is a new species. This new species has been given the binomial designation of Micromonospora peucetica sp. nova.

                                      TABLE 1                                      __________________________________________________________________________     Cultural characteristics of Strain B 211 F.I. on various media according       to Waksman, 1961,                                                              supra, where not specified otherwise.                                          Medium                Response                                                 __________________________________________________________________________     Bennett's agar        Growth good, raised and ridged, color orange to                                terra cotta                                                                    or coral red, turning to black on ageing.                Emerson's agar        Growth fair, raised and ridged. Colorless to light                             terra                                                                          cotta throughout turning brown on ageing.                Glucose-asparagine agar                                                                              Growth good, flat, color light terra cotta                                     throughout.                                              1% NZ-amine type A, 1% glycose + agar                                                                Growth good, raised and ridged. Color from orange                              terra                                                    (LUEDEMANN G.M. et al. Antimicrobial                                                                 cotta to bright red, turning brown to black on                                 ageing.                                                  Agents, 1963, pp. 116-124)                                                     Glucose-yeast extract agar                                                                           Growth poor, raised and ridged. Color light terra                              cotta                                                                          throughout.                                              Starch-casein agar    Growth good, flat. Color terra cotta throughout.         Gelatin agar          Growth poor, raised and ridged. Color terra cotta                              to                                                                             bright red.                                              Tyrosine agar (GORDON, R.D. and SMITH,                                                               Growth good, raised and ridged. Bright terra cotta       M.L., J. Bacteriol., 69, 1955, pp.                                                                   color at first, deep reddish turning copper brown                              to                                                       147-150)              almost black, on ageing. A deep brown soluble                                  pigment                                                                        is produced.                                             Yeast extract-L-tyrosine agar                                                                        Growth fair flat. Color brown, a deep reddish to                               coffee                                                                         brown soluble pigment is produced.                       Pepton-iron agar (TRESNER, H.D. and                                                                  Growth good, raised and ridged. Color light terra                              cotta                                                    DANGA, F., J. Bacteriol. 76, 1958,                                                                   throughout.                                              pp. 239-244)                                                                   Potato-glucose agar   Growth moderate, raised and ridged. Color from                                 terra                                                                          cotta to brown turning almost black on ageing.           Czapeck's agar        Growth poor, flat. Color light terra cotta turning                             black on ageing.                                         Glycerol-glycine agar Growth moderate, flat. At first colorless, turning                             olive                                                                          green to deep green on ageing.                           Inorganic-salts-starch agar                                                                          Growth fair, flat. Color from rose to terra cotta        (PRIDHAM, T.C. et al, Antibiotics                                                                    turning orange brown on ageing.                          Annual 1956/1957, pp. 947-953)                                                 Glycerol-asparagine agar                                                                             Growth poor, flat. Color light terra cotta turning                             almost black on ageing.                                  SA agar (see for composition main-                                                                   Growth good, raised and ridged. Color at first                                 creamy                                                   tenance medium in Example 1)                                                                         to honey-like turning later terra cotta and                                    becoming                                                                       slightly brownish on ageing. Sometimes a yellow                                soluble                                                                        pigment turning slightly brown on ageing is                                    observed.                                                __________________________________________________________________________

                  TABLE 2                                                          ______________________________________                                          Physiological and biochemical properties of Strain B 211                      ______________________________________                                         F.I.*                                                                          Utilization of:                                                                           glucose        +                                                               sucrose        +                                                               D-xylose       +                                                               M-mannitol     +                                                               M-inositol     +                                                               L-arabinose    +                                                               D-fructose     +                                                               Adonitol       +                                                               lactose        +                                                               d(+) mannose   +                                                               maltose        +                                                               raffinose      +                                                               L-rhamnose     +                                                               alpha-alpha-trehalose                                                                         +                                                               esculin        +                                                               glycerol       +                                                               Na-citrate     +                                                               NH.sub.4 -succinate                                                                           +                                                               Na-acetate     +                                                               NH.sub.4 -tartrate                                                                            +                                                               glycogen       +                                                               paraffin       -                                                    Negative control      -                                                        Liquefaction of gelatin                                                                              +                                                        Tyrosine decomposition                                                                               +                                                        Malanin formation     +                                                        Hydrolysis of starch  +                                                        H.sub.2 S formation   -                                                        Nitrate reduction     +                                                        Milk (pepton- and coag.)                                                       Antibiotics produced: new anthracyclines                                       ______________________________________                                          + = positive reaction                                                          - = negative reaction                                                          *The medium for the carbohydrate utilization test is as described by R.D.      Gordon and M.L. Smith, supra.                                                  *The media used for the other physiological reactions are those reported       by S.A. Waksman, 1961, supra.                                            

FERMENTATION PROCESS

The production of the new glycosides is carried out by conventional, well known methods, and comprises culturing the microorganism in a previously sterilized liquid culture medium under aerobic conditions at a temperature of from 25° C. to 37° C. (preferably at 28° C.) for from 5 to 30 days (preferably 15 days) and at a pH which initially is from 6.5 to 7.0 and which, at the end of the fermentation period is from 6.5 to 8.0.

The culture medium consists of a carbon and a nitrogen source as well as mineral salts. The carbon source may, for example, be starch, dextrin, glucose, glycerin, mannite, maltose, corn steep liquor, distillers solubles, soybean oil or soybean meal. The nitrogen source, besides those of the above-mentioned complex substances which contain nitrogen, may be for example, dry yeast, meat pepton or casein. Good results are even obtained by using ammonium salts such as ammonium nitrate, ammonium sulphates and diammonium phosphates. The mineral salts vary according to the medium employed. In a medium containing complex substances such as various meals and fermentation residues, the addition of calcium carbonate and sodium or potassium phosphates has proved useful. In media containing glucose, or ammonium salts, much higher levels of mineral salts such as potassium, sodium or calcium salts, and additions of trace elements such as iron, zinc, copper, magnesium and manganese are necessary. The fermentation may be carried out in Erlenmeyer flasks or in laboratory or industrial fermenters of various capacities.

ANALYTICAL METHODS

Samples of the fermentation broth and crude preparations are subjected to paper chromatography using Whatman No. 1 paper, buffered with M/15 phosphate buffer at pH 5.4, employing as the eluant a mixture of n-propanol:ethyl acetate:water (7:1:2). The paper strips are bioautographed against Bacillus subtilis, and four antibiotic components were found to occur. These are designated glycoside A (Rf 0.30), B (Rf 0.50), C (Rf 0.55) and D (Rf 0.65).

Crude preparations are subjected to thin layer chromatography (TLC) using pre-coated TLC plates of silica gel 60-F-254 (Merck) and as the eluant, a mixture of chloroform:methanol:acetic acid:water (80:20:14:6). Four yellow compounds occur, and they correspond to glycoside A (Rf 0.50), B (Rf 0.55), C (Rf 0.65) and D (Rf 0.70) respectively. A quantitative estimation of the total yellow constituents present in the fermentation broth can be made by the following method. To a sample of broth, adjusted to pH 8.6, two volumes of chloroform:methanol (9:1) are added. The resulting mixture is sonicated for 1 minute at room temperature. On a sample of the organic phase, diluted with acidic methanol, the total content of the yellow anthracyclines and of their aglycones can be spectrophotometrically determined at 418 nm. On a sample of the organic phase, concentrated under reduced pressure, quantitative determination of the single glycosides can be obtained by preparative TLC using the above reported system. The different yellow zones are scraped off and eluted with methanol. Each constituent is spectrophotometrically determinated at 418 nm. Glycoside A is usually the major constituent in the fermentation broths.

ISOLATION PROCEDURE

After the fermentation, the active compounds are contained in the mycelia and in the fermentation liquor. The anthracycline antibiotic complex can be extracted at pH 8.5-9.0 in the form of the free bases from the culture broth "in toto" with a water immiscible organic solvent such as butanol, methyl isobutyl ketone, chloroform, methylene dichloride or ethyl acetate. Preferably, the mycelia and the fermentation liquor are separated by filtration at pH 4 with the aid of diatomaceous earth, and then extracted separately.

The filtration cake is extracted with a mixture of a water-soluble solvent, such as acetone, methanol or other lower alcohol, and an 0.1 N aqueous solution of an inorganic or organic acid, such as hydrochloric acid, sulphuric acid or acetic acid. Generally, a mixture of acetone: 0.1 N hydrochloric acid in a ratio 4:1 by volume is employed. The mycelia extracts are collected, adjusted to pH 4, and then concentrated under reduced pressure. The aqueous concentrate is combined with the filtered broth, adjusted to pH 8.5-9.0, then extracted with a water immiscible organic solvent, preferably chloroform or n-butanol. The extracts are concentrated under reduced pressure and the anthracycline complex is precipitated by addition of five volumes of n-hexane. The constituents of the crude complex are then fractionated and purified by column chromatography.

PURIFICATION PROCEDURE

Further purification of the antibiotic complex and its separation into its four components may be effected by silica gel column chromatography. The crude orange brown powder is dissolved in chloroform and the solution, mixed with an equivalent of methanolic hydrogen chloride, is chromatographed on silica gel with chloroform:methanol:water mixtures. The components D and C are eluted first, with a 94.8:5.0:0.2 mixture. The glycosides B and A follow with an 89.5:10.0:0.5 mixture. The components are usually separated as shown by paper and thin layer chromatography and the four components are obtained as their hydrochlorides in crystalline form.

CHEMICAL AND PHYSICAL PROPERTIES

The novel antibiotics of the invention have some common properties, but they can be distinguished on the basis of their chemical and physical characteristics. All the new anthracyclines have comparable solubility; as free bases they are soluble in chloroform, methylene dichloride, acetone, methanol, ethanol, aqueous alcohols, acidic water, dioxane and pyridine, but sparingly soluble or insoluble in diethyl ether, n-hexane, cyclohexane and petroleum ether. As hydrochlorides, they are soluble in water, methanol, ethanol and aqueous alcohols, but insoluble in acetone, benzene, chloroform, diethyl ether and pertroleum ether. They can be used as indicators, being orange-yellow in neutral and acidic solutions, in which they also show orange-red fluorescence under U.V. light. Their alkaline solutions are red-brown, and when treated with alcoholic magnesium acetate they form red solutions. All these properties and the absorption spectra in the ultraviolet and visible regions, indicate that these new compounds are anthracycline antibiotics. The chemical and physical properties of glycosides A, B, C and D, isolated as the hydrochlorides are given in Table 3.

                                      TABLE 3                                      __________________________________________________________________________     Chemical and physical properties of Glycosides A, B, C and D                   PROPERTY                                                                               GLYCOSIDE A. HCl                                                                           GLYCOSIDE B. HCl                                                                           GLYCOSIDE C. HCl                                                                           GLYCOSIDE D. HCl                   Melting Point                                                                          171°-173° (dec.)                                                             163°-164° (dec.)                                                             175°-176° (dec.)                                                             140°-150°            __________________________________________________________________________                                                 (dec.)                             [α] 23°                                                           (c = 0.2 in                                                                    methanol)                                                                              +111°                                                                               +107°                                                                               +139°                                                                               +122°                       U.V. and                                                                       Visible Spectra                                                                λMeOH max                                                                       228,260,418 nm                                                                             228,260,418 nm                                                                             228,260,418 nm                                                                             228,260,418 nm                      1%                                                                             1 cm   645,420,193 640,410,179 713,450,199 610,395,171                        λpH 7 buffer max                                                                235,262,426 nm                                                                             235,262,246 nm                                                                             235,262,426 nm                                                                             235,262,426 nm                      1%                                                                            E                                                                               1 cm   600,406,161 605,400,160 617,424,166 580,400,160                        λ0.01 N NaOH max                                                                510 nm      510 nm      510 nm      510 nm                              1%                                                                            E                                                                               1 cm   120         118         128         110                                I.R. Spectrum                                                                          3,700-2,400                                                                             1,200                                                                             3,700-2,400                                                                             1,195                                                                             3,700-2,300                                                                             1,195                                                                             3,700-2,400                                                                             1,19                      (KBr):cm.sup.-1                                                                        1,725    1,180                                                                             1,665    1,180                                                                             1,710                1,18                              1,670    1,115                                                                             1,625    1,115                                                                             1,670    1,115                                                                             1,665    1,11                              1,625    1,085                                                                             1,585       1,625       1,625    1,04                              1,585    1,055                                                                             1,485    1,050                                                                             1,585    1,085                                                                             1,585    1,01                              1,490    1,015                                                                             1,470    1,010                                                                             1,490    1,010                                                                             1,485    98                                1,470    985                                                                               1,445    985                                                                               1,470    985                                                                               1,465    83                                1,450    940                                                                               1,420    940                                                                               1,445    835                                                                               1,445    75                                1,420    875                                                                               1,385    840                                                                               1,420    815                                                                               1,420    43                                1,385    840                                                                               1,325    820                                                                               1,385    795                                                                               1,385                                      1,330    820                                                                               1,290    750                                                                               1,325    750                                                                               1,325                                      1,300    795                                                                               1,235    435                                                                               1,295    435                                                                               1,295                                      1,290    755            1,235       1,255                                      1,240    440                        1,235                              Empirical                                                                              C.sub.27 H.sub.29 NO.sub.10 . HCl                                                          C.sub.27 H.sub.31 NO.sub.9 . HCl                                                           C.sub.27 H.sub.29 NO.sub.9 .                                                               C.sub.27 H.sub.31 NO.sub.8 .                                                   HCl                                Formula                                                                        Molecular                                                                      Weight  564         550         548         534                                __________________________________________________________________________

The new anthracycline antibiotics also show characteristic proton magnetic resonance (PMR) spectra. The spectrum of glycoside A, as the hydrochloride in DMSO-d₆, showed characteristic signals at 1.14δ (d, CH₃ --C--5'), 3.39δ (s, CH₃ O), 4.60δ (broad s, C--14--H₂) 4.89δ (broad s, C--7--H), 5.27δ (broad s, C--1'--H), 7.23δ (s, C--11--H), 7.4-7.9δ (m, C--1--H, C--2--N and C--3--H) and 13.61δ (s, C--6--OH) glycoside C, as the hydrochloride in DMSO-d₆, showed signals at 1.15δ (d, CH₃ --C--5'), 2.26δ (s, CH₃ CO), 3.92δ (s, CH₃ O), 4.90δ (broad s, C--7--H), 5.26δ (broad s, C--1'--H) 7.31δ (s, C-- 11--H), 7.4-7.9δ (m, C--1--H, C--2H, and C--3--H) and 13.65δ (s, C--6--OH).

Field desorption mass spectroscopy of the free bases of glycosides A, B, C and D confirmed the assigned molecular formulae as shown in Table 4.

                  TABLE 4                                                          ______________________________________                                                            Molecular Weight                                            Compound   Molecular Formula                                                                            Calculated                                                                               Found: m/e                                  ______________________________________                                         GLYCOSIDE A                                                                               C.sub.27 H.sub.29 NO.sub.10                                                                  527       527 (M.sup.+)                               GLYCOSIDE B                                                                               C.sub.27 H.sub.31 NO.sub.9                                                                   513       514 (MH.sup.+)                              GLYCOSIDE C                                                                               C.sub.27 H.sub.29 NO.sub.9                                                                   511       512 (MH.sup.+)                              GLYCOSIDE D                                                                               C.sub.27 H.sub.31 NO.sub.8                                                                   497       498 (MH.sup.+)                              ______________________________________                                    

Aqueous acid hydrolysis of glycosides A, B, C and D gives four different water insoluble yellow aglycones of the formulae V-VIII, above. The aqueous soluble fractions all contained the same reducing amino-sugar which was identified as daunosamine (3-amino-2,3,6-trideoxy-L-lyxohexose), the amino sugar component of daunomycin and adriamycin.

The structures of the aglycones were determined by analysis of their infrared, ultraviolet, visible, mass and magnetic resonance spectra. Their chemical and physical properties are reported in Table 5.

                                      TABLE 5                                      __________________________________________________________________________     Chemical and physical properties of the aglycones of glycosides A, B, C        and D                                                                          PROPERTY  AGLYCONE OF A                                                                             AGLYCONE OF B                                                                             AGLYCONE OF C                                                                             AGLYCONE OF D                       __________________________________________________________________________     Melting point °C.                                                                 220        175-180 with                                                                              213-215    175-180 with                                             decomposition         decomposition                       [α].sub.D.sup.23° (c = 0.1)                                                 +161°          +144°                                                                              +164°                                  (dioxan)              (methanol) (methanol)                          U.V. e VIS Spectra                                                             γMeOH max                                                                          227,259,418 nm                                                                            227,258,418 nm                                                                            227,258,418 nm                                                                            227,258,418 nm                       1%                                                                            E 1 cm    895,640,268           930,660,317                                                                               990,640,280                         Empirical formula                                                                        C.sub.21 H.sub.18 O.sub.8                                                                 C.sub.21 H.sub.20 O.sub.7                                                                 C.sub.21 H.sub.18 O.sub.7                                                                 C.sub.21 H.sub.20 O.sub.6           Molecular weight                                                               calculated                                                                               398        384        382        368                                 Found m/e 398 (M.sup.+)                                                                             384 (M.sup.+)                                                                             382 (M.sup.+)                                                                             368 (M.sup.+)                       __________________________________________________________________________

BIOLOGICAL ACTIVITY DATA

(a) Antibacterial activity

The "in vitro" minimum inhibitory concentrations (MIC) of glycosides A, B, C and D were determined for certain microorganisms using the standard tube dilution procedure and are reported in Table 6.

                  TABLE 6                                                          ______________________________________                                                   MIC in μg/ml                                                                Glycosides                                                           Test Organism                                                                              A (I)    B (II)   C (III)                                                                               D (IV)                                    ______________________________________                                         Staphylococcus                                                                             125      1000     62     250                                       aureus 209 P                                                                   Staphylococcus                                                                             500      1000     250    1000                                      aureus 153                                                                     Sarcina lutea                                                                              100      100        12.5  25                                       ATCC 9341                                                                      Bacillus subtilis                                                                          100      100      50     100                                       ATCC 6633                                                                      Escherichia coli B                                                                          50      100      25      50                                       ______________________________________                                    

(b) Antitumor activity

The new glycosides were tested against HeLa cells in vitro (time of exposure to the drugs: 24 hours), and on L 1210 and P 388 leukemia in mice in comparison with daunomycin. The results of the in vitro tests are given in Table 7. It can be seen therefrom that all the compounds inhibited the cell viability of HeLa cells in vitro, the ID₅₀ being 0.05 μg/ml for glycoside C, 0.1 μg/ml for glycoside A, 0.22 μg/ml for glycoside D and 0.44 μg/ml for glycoside B.

The in vivo data obtained in mice are reported in Table 8. Glycoside A was active against P 388 leukemia at a dose of 66 mg/kg, and toxic at a dose of 100 mg/kg. At the tolerated dose, the antitumor activity was greater than that of daunomycin.

                  TABLE 7                                                          ______________________________________                                         Effect on HeLa cells viability in vitro                                                  Dose(a)   No. of colonies(b)                                                                           ID.sub.50                                    Compound  (ng/ml)   %             (ng/ml)                                      ______________________________________                                         Daunomycin                                                                               12.6      35-15          6-7                                                   6.25      48-66                                                                3.12      71-80                                                      Glycoside A                                                                              200       1                                                          (I)       100       63             105                                                   50        73                                                                   25        74                                                         Glycoside C                                                                              200       3                                                          (III)     100       44             54                                                    50        54                                                         Glycoside D                                                                              400       0                                                          (IV)      200       53             220                                                   100       109                                                        Glycoside B                                                                              1600      0                                                          (II)      800       20             440                                                   400       66                                                         ______________________________________                                          (a)HeLa cells were exposed to the drugs for 24 hours, then plated. Number      of colonies was evaluated 5 days later.                                        (b)% over untreated controls.                                            

                  TABLE 8                                                          ______________________________________                                         Effect against P 388 and L 1210 leukemia in mice                                       Dose (a)                                                                               T/C%           Toxic (b)                                       Compound  (mg/kg)   L 1210    P 388  deaths                                    ______________________________________                                         Daunomycin                                                                               2.9       133-133   190                                                        4.4       140-133   200                                                        6.6       111-133   223    14/26                                     Glycoside A                                                                              2.9       111                                                        (I)       6.6       128                                                                  10        122                                                                  66                  245                                                        100                 254     1/10                                     Glycoside C                                                                              2.9       100                                                        (III)     6.6       117                                                                  15        122                                                                  100                 181    3/8                                                 150                  45     3/19                                     Glycoside D                                                                              2.9       100                                                        (IV)      6.6       100                                                                  15        111                                                                  125                  18    5/5                                                 250        0         9     5/5                                       Glycoside B                                                                              2.9       100                                                        (II)      6.6       122                                                                  15        122                                                                  500                  9     2/2                                       ______________________________________                                          (a)Mice were treated intraperitoneally on day 1 after tumor cell               inoculation.                                                                   (b)Evaluated on the basis of the macroscopic autopsy findings.           

The following Examples are further illustrative of the invention.

EXAMPLE 1

A culture of Micromonospora peucetica, strain B 211 F.I., was grown for 25 days at 28° C. on agar slants of maintenance medium SA. Medium SA has the following composition: glucose 3%; brewers dry yeast 1.2%; sodium chloride 0.1%; potassium dihydrogen orthophosphate 0.05%; calcium carbonate 0.1% magnesium sulphate 0.005%; ferrous sulphate heptahydrate 0.0005%; zinc sulphate heptahydrate 0.0005%; cupric sulphate pentahydrate 0.0005%; agar 2%; tap water up to 100%. Sterilization was carried out by heating in an autoclave at 120° C. for 20 minutes.

The mycelial mat of the culture so obtained was scraped off and suspended in 3 ml of sterile distilled water. The suspension was inoculated in 300 ml Erlenmeyer flasks containing 60 ml of the following liquid growth medium: brewers dry yeast 0.3%; peptone 0.5%; calcium nitrate tetrahydrate 0.05%; tap water up to 100%. Sterilization was effected by heating in an autoclave at 120° C. for 20 minutes. The pH of the medium after sterilization was between 6.8 and 7.0.

The inoculated flasks were shaken for 8 days at a temperature of 28° C. on a rotary shaker running at 250 rpm and describing a circle of 7 cm in diameter. 5 ml of the culture grown as described above were inoculated in a 300 ml Erlenmeyer flask containing 50 ml. of the following production medium: glucose 6%; brewers dry yeast 3%; sodium chloride 0.2%; potassium dihydrogen orthophosphate 0.1%; calcium carbonate 0.2% magnesium sulphate 0.01%; ferrous sulphate heptahydrate 0.001%; zinc sulphate heptahydrate 0.001%; cupric sulphate pentahydrate 0.001%; tap water up to 100%; sterilization by heating in autoclave at 115° C. for 20 minutes.

The flasks were incubated at 28° C. for 25 days under conditions identical to those described for the seed phase. The maximum concentration of the active compounds was reached between the 18th and 22nd days of fermentation with a production of 90 mcg/ml.

EXAMPLE 2

A culture of strain B 211 F.I. was grown for 14 days as described in Example 1, and the mycelial mat, collected as described in Example 1, was inoculated in 300 ml Erlenmeyer flasks containing 50 ml of the following liquid growth medium: soluble starch, 4%; soy bean flour 1.5%; brewer's dry yeast 0.5%, corn steep liquor 0.8%; calcium carbonate 0.3%; potassium monohydrogen orthophosphate 0.05%; magnesium sulphate 0.025%; potassium chloride 0.025%; tap water up to 100%. The pH, being 5.7 was brought to 7.5 with sodium hydroxide before sterilization. Sterilization was effected at 120° C. for 20 minutes.

After 4 days of incubation under the same conditions as described in Example 1, 5 ml of the thus grown culture were inoculated in 300 ml Erlenmeyer flasks containing 50 ml of the medium described above.

The flasks were then incubated under the same conditions as described above for 10 days. The maximum concentrations of the active compounds was reached between the 8th and 9th days of fermentation with a production of 15 mcg/ml.

EXAMPLE 3

The whole beer (20 liters) from a fermentation broth obtained according to Example 1, was adjusted to a pH of about 4 with hydrochloric acid and filtered, using 3% diatomaceous earth as a filter aid, to yield a cake and a filtrate which were extracted separately. The wet filter cake was treated with about 4 liters of a mixture of acetone and 0.1 N aqueous hydrochloric acid (80:20). After filtration, a second treatment with an additional 3 liters of acidified aqueous acetone assured complete extraction of the active compounds. The combined aqueous acetone extracts were adjusted with ammonium hydroxide to a pH of about 4, concentrated under reduced pressure to about 1 liter, and combined with the filtered broth. The obtained mixture was adjusted to a pH of about 8.5 to 9.0 and then extracted twice with one-half volume of a chloroform:methanol (9:1) mixture. The combined organic extracts were washed with water, dried on anhydrous sodium sulphate, and concentrated under reduced pressure to a volume of about 200 ml. By addition of 1 liter of n-hexane, the crude complex precipitated as a yellow brown powder (7 g).

EXAMPLE 4

A chloroform solution (6 g in 200 ml) of the crude complex (free base) after treatment with 10 ml of 1 N methanolic hydrogen chloride, was placed on a column of silicic acid (prepared in chloroform). The column was washed with chloroform, followed by elution with a chloroform:methanol:water mixture (94.8:5:0.2). The first yellow fraction contained some aglycones. The next two yellow bands contained, respectively, glycosides D and C. Elution was continued with a chloroform:methanol:water mixture (89.5:10:0.5) until two other yellow bands, corresponding to glycosides B and A, were eluted. The fractions containing these four bands were separately concentrated under reduced pressure to give substantially pure hydrochlorides of glycoside D (0.2 g), glycoside C (0.4 g), glycoside B (0.2 g) and glycoside A (0.6 g) as microcrystalline powders. Recrystallization of the glycosides A and C from methanol:n-butanol gave the corresponding pure hydrochlorides as yellow-orange crystals, m. p. 171°-173° C. with decomposition for glycoside A, m.p. 163°-164° C. with decomposition for glycoside B, m.p. 175°-176° C. with decomposition for glycoside C and m.p. 140°-150° C. with decomposition for glycoside D.

EXAMPLE 5

A 250 mg sample of glycoside A was dissolved in 10 ml of 0.2 N aqueous hydrochloric acid and the solution was heated for 1 hour at 95° C. A crystalline yellow-orange precipitate was collected by filtration after cooling, washed with water and dried over phosphorus pentoxide overnight under vacuum. 170 mg of the aglycone of glycoside A were obtained in pure form, m.p. 220° C., m/e: 398 (M⁺). After precipitation of the aglycone, the almost colorless aqueous acidic solution was adjusted to pH 5 with an anion exchange resin and then freeze-dried. The residue (60 mg), crystallized from methanol:acetone gave a crystalline compound, m.p. 166° C. with decomposition, identified as daunosamine hydrochloride by direct comparison with an authentic sample. Similar treatment of glycosides B, C and D yield the respective agylcones thereof.

Variations and modifications can, of course, be made without departing from the spirit and scope of the invention. 

Having thus described our invention, what we desire to secure by Letters Patent and hereby claim is:
 1. A compound having the formula: ##STR4## wherein R is --CO--CH₂ OH, --CHOH--CH₃, --CO--CH₃ or --CH₂ --CH₃ and a salt thereof with a pharmaceutically acceptable acid or metallic cation.
 2. A compound according to claim 1 which is 11-deoxyadriamycin.
 3. A compound according to claim 1 which is 11-deoxy-13-dihydro-daunomycin.
 4. A compound according to claim 1 which is 11-deoxydaunomycin.
 5. A compound according to claim 1 which is 11-deoxy-13-deoxo-daunomycin.
 6. A pharmaceutical composition comprising a therapeutically effective amount of a compound as claimed in claim 1 and an inert carrier therefor. 